Combination of 25-hydroxyvitamin d and antioxidants/anti-inflammatories for human nutraceuticals

ABSTRACT

This invention relates to the use of the combination of 25-hydroxyvitamin D3 (“25-OH D3”) and antioxidants/anti-inflammatories (ascorbic acid, vitamin E and at least one carotenoid) to make a pharmaceutical, nutraceutical or food supplement which can ameliorate various problems observed in humans connected with polycystic ovarian 5 syndrome and cardiovascular diseases. Pharmaceuticals, nutraceuticals and food supplements containing the 25-OH D3 and antioxidants/anti-inflammatories and premixes are also provided.

This application claims priority from U.S. provisional application62/103,769 filed 15 Jan. 2015, and EP15166937.1 filed 8 May 2015, bothof which are hereby incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to the combination of 25-hydroxyvitamin D (“25-OHD3” and/or “25-OH D2”) and antioxidants/anti-inflammatories (ascorbicacid, Vitamin E and a carotenoid) for use in human nutraceuticals,pharmaceuticals and food supplements. This combination of nutritionalsupplements protects against various adverse effects associated withhyperphagia and related obesity. It also relates to nutraceuticals,nutraceutical premixes, pharmaceuticals and food containing thecombination of 25-hydroxyvitamin D and theantioxidants/anti-inflammatories.

BACKGROUND OF THE INVENTION

Hyperphagia (overeating) and its related obesity can cause variousproblems in humans. These problems include: ovarian dysfunction andinfertility as well as metabolic disorders related to the cardiovascularsystem and the blood sugar system.

Interestingly, one animal model for female ovarian disease is poultry.The domestic laying hen is the only non-human animal that developsovarian cancer with a high prevalence. See Johnson et al 2013 Nature 13:432-436, and Walzem et al 2014 Advances in Nutrition 5: 199-206. Theprogression of hen ovarian cancer as well as locations of metastaticgrowths and ascites are similar to that observed in women.

Some breeds of poultry experience hyperphagia-related obesity.Generally, the farmer will restrict the amount of food offered to theflock to prevent the adverse consequences of obesity. However, it isoften difficult to determine the proper amount of feed to provide whichwill ensure the desired growth of the flock, and under restrictedfeeding conditions, individuals can become aggressive and not onlyinjure other birds, but become obese themselves.

Female broiler breeders overfed during reproductive development not onlyproduce excess large yellow ovarian follicles but also generate agreater number of atretic yellow follicles and commonly display erraticoviposition and defective egg syndrome (EODES) that include severalreproductive problems such as follicular atresia, the production ofsoft-shelled or membranous eggs, double-yolked eggs, egg yolkperitonitis (presence of egg yolk in the abdominal cavity), multiple eggdays and oviposition not occurring in sequence, resulting in increasedproduction of unsettable eggs.

Controlled studies reported that voluntary feeding (i.e., broilerbreeder hens fed to satiation) resulted in poor egg production, highrate of mortality and abnormal ovarian structure (mainly overthierarchical follicle atresia). In contrast to feed-restricted hens,voluntary feeding also induced metabolic dysregulations that comprisedenhanced adiposity; hepatic triacylglycerol accumulation; and elevatedconcentrations of plasma glucose, non-esterified fatty acids, very lowdensity lipoprotein, triacylglycerol, phospholipids, ceramide andsphingomyelin.

Furthermore, hepatic and circulating ceramide and sphingomyelinaccumulation, and up-regulation of proinflammatory IL-1β expression inliver and adipose tissues systemically manifested the development oflipotoxicity in feed-satiated hens. Ceramide is a key intermediatelinking certain nutrients (i.e. saturated fats) and inflammatorycytokines (e.g. tumor necrosis factor-α, TNFα) to the regulation of cellfunction and antagonizing insulin signaling and mitochondrial function.Moreover, as a result of its toxic effects on particularly susceptiblecell types, ceramide has the capacity to damage the heart, pancreas, andvasculature. Lipotoxicity leading to impaired ovarian dysfunctions,including follicle atresia, ovarian regression, and a decline ofcirculating estradiol levels in feed-satiated hens, was furtherexemplified by ceramide accumulation and up-regulation of IL-1β, serinepalmitoyltransferase, and sphingomyelinase transcript abundance, butsuppressed protein kinase Akt activation within the hierarchicalfollicles. In vivo evidence has thus delineated the actions of ceramideand IL-1β in mediating overfeeding-induced follicle atresia andprogression of ovarian involution in broiler hens.

Despite restricted feeding regimen strictly implemented in commercialbroiler breeder flocks, it is still very easy to overfeed breeder hensdue to their intrinsic hyperphagia. Furthermore, breeder farm managersare confronted as to when and how to feed before and during the start ofegg production as well as towards, during and after peak production. Thebasic fundamental question to ask what and how management andnutritional tools breeder farm managers can apply and implement toameliorate the adverse and deleterious effects of reproductiveefficiency associated with obesity in overweight hens.

Hy⋅D® (registered trademark for 25-OH-D3; available from DSM NutritionalProducts, Switzerland) has been used to promote bone health in poultry,swine, and for vitamin D deficiency in humans.

The combination of 25-OH D3 and canthaxanthin has also been used inpoultry. WO2010/057811 (DSM IP ASSETS, BV) describes this combinationfor use in improving hatchability, fertility, and lower embryo mortalityin poultry. The combination is commercially available under thetrademark MAXICHICK. There is no mention in the patent publication ofthe inclusion of ascorbic acid and high vitamin E levels, nor the usesto ameliorate the adverse effects of hyperphagia-related obesity.

Vitamin C (ascorbic acid) is often not included as a supplement inpoultry diets, as the chicken can under normal rearing conditions canproduce sufficient Vitamin C. However, it has been used in some specificconditions, such as in heat stress situations.

Vitamin E is generally added to poultry feed. Recommended doses forpoultry species tends to range from about 50-100 IU/kg feed, dependingon the age of the animal.

WO14/202433 (DSM IP ASSETS B.V) teaches the combination of canthaxanthinand 25-OH D3 to improve internal egg quality, i.e. enhancing thestrength of vitelline membrane that envelops the yolk. There is noteaching to add ascorbic acid to the combination, nor for its use inameliorating the adverse effects of hyperphagia-related obesity.

WO14/191153 (DSM IP ASSETS B.V) teaches the combination of canthaxanthinand at least one of Vitamin C, Vitamin E, selenium, and optionally atleast one of thymol, eugenol, vanillin and gamma-terpinene can improveimmune statues, bone health, skeletal development and growth and feedconversion, particularly when flocks are subject to stress associatedwith vaccination.

There is a need to prevent or delay the onset of ovarian diseases andcardiovascular related problems in humans, as well as to modulate weightgain associated with hyperphagia.

DETAILED DESCRIPTION OF THE INVENTION

It has been found, accordance with this invention that the combinationof 25-hydroxyvitamin D (25-OH D3 and/or 25-OH D2) andantioxidants/anti-inflammatories can benefit ovarian health, andparticularly polycystic ovarian syndrome. In addition to benefitingovarian health, the combination of 25-OH D andantioxidants/anti-inflammatories can benefit cardiovascular andmetabolic health and modulate weight gain in people who overeat. It hasalso been found in accordance with this invention, that the bio-activestraditionally thought of as antioxidants also possess ananti-inflammatory activity which benefits ovarian and cardiovascularhealth.

As 25-OH D2 and 25-OH D3 may act in a similar fashion afteradministration, it is envisioned that either may be used separately incombination with antioxidants/anti-inflammatories, or a mixture of both25-OH D3 and 25-OH D2 may be used in combination withantioxidants/anti-inflammatories. If used together, the ratio of 25-OHD3: 25-OH D2 is not a critical part of the invention. 25-OH D3 usedalone is preferred.

The antioxidants/anti-inflammatories of this invention comprise thecombination of ascorbic acid, vitamin E and a carotenoid. The carotenoidis at least one selected from the group consisting of: lycopene,astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin andcanthaxanthin. Most preferred are lycopene, astaxanthin, lutein, andzeaxanthin. Thus one aspect of this invention is the combination of25-OH D3, one or more of the aforementioned carotenoids, vitamin E andascorbic acid. Another embodiment is the combination of 25-OHD2, one ormore of the aforementioned carotenoids, Vitamin E and ascorbic acid.Another embodiment is the combination of 25-OHD3, 25-OHD2, one or moreof the aforementioned, Vitamin E and ascorbic acid.

Compositions

Another aspect of this invention is the combination of 25-OH D, one ormore of the aforementioned carotenoids, Vitamin E and ascorbic acid,which optionally further comprises at least one further bio-activeingredient selected from the group consisting of:

-   -   Vitamin D, Vitamin B2, Biotin, Vitamin B6, Niacin, Zinc, Copper,        Manganese, and Selenium. Preferably the 25-OH D is 25-OH D3.        Preferably at least Vitamin D is a further bio-active        ingredient. Sometimes the further bio-active ingredients include        at least Vitamin D and Selenium. In some cases, all the further        bio-active ingredients are added.

A further aspect is the combination of 25-OH D, one or more carotenoidsof this invention, vitamin E and ascorbic acid which optionally furthercomprises at least one further bio-active ingredient selected from thegroup consisting of Vitamin D, Vitamin B2, Vitamin B6, Niacin,Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, Selenium,and combinations thereof. Preferably the 25-OH D is 25-OH D3. Sometimes,the further bio-active ingredient includes biotin. Sometimes the furtherbio-active ingredient includes Vitamin D and biotin. Sometimes thefurther bio-active ingredient includes all the aforementioned optionallybio-active ingredients.

Nutraceuticals, Pharmaceuticals, Dietary Supplements

Another aspect of this invention is a nutraceutical such as a dietarysupplement, or pharmaceutical comprising the combination of 25-OH D2 or25-OH D3 or mixtures thereof, ascorbic acid, Vitamin E and one or moreof the aforementioned carotenoids.

Yet another embodiment is a nutraceutical such as a dietary supplement,or a pharmaceutical comprising the combination of 25-OH D, one or moreof the aforementioned carotenoids, vitamin E and ascorbic acid, whichoptionally further comprises at least one further bio-active ingredientselected from the group consisting of: Vitamin D, Vitamin B2, VitaminB6, Niacin, Zinc, Copper, Manganese, Selenium and combinations thereof.Preferably the 25-OH D is 25-OH D3. Sometimes the further bio-activeingredients include at least Vitamin D and Selenium. In some cases, allthe further bio-active ingredients are added.

Another embodiment is a nutraceutical, such as a dietary supplement or apharmaceutical comprising the combination of 25-OH D, one or more of theaforementioned carotenoids, vitamin E and ascorbic acid, whichoptionally further comprises at least one further bio-active ingredientselected from the group consisting of: Vitamin D, Vitamin B2, VitaminB6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper,Manganese, Selenium and combinations thereof. Sometimes, the furtherbio-active ingredient includes biotin. Preferably the 25-OH D is 25-OHD3. Sometimes the further bio-active ingredient includes Vitamin D andbiotin. Sometimes the further bio-active ingredient includes all theaforementioned optionally bio-active ingredients.

Premixes

Another aspect of this invention are premixes for nutraceuticals, suchas food supplements or vitamin supplements which comprise thecombination of 25-OH D, vitamin E, ascorbic acid and one or more of theaforementioned carotenoids. Preferably, the 25-OH D is 25-OH D3. Thepremixes and subsequent nutraceutical improves/ameliorates adverseconditions associated with the ovaries, and specifically polycysticovarian syndrome. In some embodiments the premix or subsequentnutraceutical also comprises at least one further bio-active ingredientselected from the group consisting of:

-   -   Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper,        Manganese, Selenium and combinations thereof. Sometimes the        further bio-active ingredients include at least Vitamin D and        Selenium. In some cases, all the further bio-active ingredients        are added.

Another aspect of this invention are premixes for nutraceuticals whichcomprise which further comprise at least one further bio-activeingredient selected from the group consisting of: Vitamin D, Vitamin B2,Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper,Manganese, Selenium and combinations thereof. Sometimes, the furtherbio-active ingredient includes biotin. Sometimes the further bio-activeingredient includes Vitamin D and biotin. Sometimes the furtherbio-active ingredient includes all the aforementioned optionallybio-active ingredients.

When using the nutraceuticals or pharmaceuticals of this invention, theperson may eat normally or over-eat, and the ill effects which affectthe ovaries, cardiovascular system and metabolic system and are normallyassociated with eating a non-calorie restricted diet will be experiencedto a lesser degree or not at all.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows glucose clearance and insulin secretion of broiler breederhens in response to ad libitum feed intake in combination with 25-OHD3+antioxidants/anti-inflammatories. Hens were injected with a singledose of glucose (0.5 g/kg BW) through wing vein after 3 weeks offeeding. Blood samples were collected through cannulation of wing veinat indicated time points after glucose infusion, n=3.

FIG. 2 shows tissue interleukin-1β contents and plasma IL-6 levels ofbroiler breeder hens in response to ad libitum feed intake incombination with 25-OH D3 and antioxidants/anti-inflammatories. Tissuesand blood samples were collected after 10 weeks of the feeding trial.Means with different superscript letters are significantly different(P<0.05), n=3.

FIG. 3. shows tissue STAT-3 activation of broiler breeder hens inresponse to ad libitum feed intake in combination with 25-OH D3 andantioxidants/anti-inflammatories inclusion. Tissues and blood sampleswere collected after 10 weeks of the feeding trial. Means with differentsuperscript letters are significantly different (P<0.05), n=3.

FIGS. 4-6 show pictures taken during the necropsy of broiler hens inresponse to ad libitum feed intake in combination with 25-OH D3 andantioxidants/anti-inflammatories inclusion. Hens were necropsied after10 weeks of the feeding trial.

FIG. 7 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on egg production of broilerhens with restricted or ad libitum feed intake.

FIG. 8 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on the survival rate ofbroiler hens with restricted or ad libitum feed intake.

FIG. 9 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on body weight of broiler henswith restricted or ad libitum feed intake.

FIG. 10 is a series of photos showing the gross morphology of the heartof dead hens with dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitumfeed intake.

FIG. 11 is a series of photographs showing the gross morphology of theheart of dead hens with dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitumfeed intake

FIG. 12 is a series of electrocardiograms (EGCs)demonstrating the effectof dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatorieson EGC of broiler hens with restricted or ad libitum feed intake. Thearrows point to irregularities in the patterns.

FIG. 13 is a series of EGSs showing the effect of dietarysupplementation of 25-OH-D3+antioxidants/anti-inflammatories onarrhythmic ECG pattern of broiler hens with restricted or ad libitumfeed intake.

FIG. 14 shows photos and a graph demonstrating the effect of dietarysupplementation of 25-OH-D3+antioxidants/anti-inflammatories on cardiacfibrosis of broiler hens with restricted or ad libitum feed intake. (atage of 35 weeks)

FIG. 15 are graphs showing the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on plasma IL-6 and IL-1βconcentration of broiler hens with restricted or ad libitum feed intake.

FIG. 16 are photos showing the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on cardiac cell apoptosis ofbroiler hens with restricted or ad libitum feed intake. (at age of 35weeks)

As used throughout this specification and claims, the followingdefinitions apply: “25-OH D” refers to any form of 25-hydroxyvitamin D(i.e. either 25-OH D2 or 25-OH D3, or mixes thereof). 25-OH D3specifically refers to 25-hydroxyvitamin D3; 25-OH D2 specificallyrefers to 25-hydroxyvitamin D2.

“Vitamin D” means either Vitamin D2, Vitamin D3 or a combination.Vitamin D3 used alone is preferred.

“Hyperphagia” is excessive eating; the person's intake of calories isgreater than their energy expenditure.

“Ameliorating weight gain” means that there is a significantly loweramount of weight gain when eating ad libitum while ingesting thecombination of 25-OH D and antioxidants/anti-inflammatories describedherein. There may be a positive weight gain compared to eating a caloriecontrolled diet.

“Ascorbic Acid” and “Vitamin C” are used interchangeably throughout thespecification and claims.

“Basal diet” means that the feed supplies the poultry with sufficientvitamins and minerals so that the poultry are vitamin and mineralreplete.

“25-OH-D3+antioxidants/anti-inflammatories” means the combination of25-OH D3, vitamin E, carotenoids of this invention (see next paragraph)and ascorbic acid, administered in addition to a diet which providessufficient vitamins and minerals so that the person is not suffering avitamin or mineral deficiency or insufficiency, at a dosage range as setforth in the specification. Optionally, and preferably, additionalbio-active ingredients, selected from the group consisting of: VitaminD, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin,Zinc, Copper, Manganese, Selenium and combinations thereof are added tothe 25-OHD3, vitamin E, canthaxanthin and ascorbic acid combination.

“Carotenoids of this invention” means at least one of: lycopene,astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin andcanthaxanthin. Most preferred are at least one of: lycopene,astaxanthin, lutein, and zeaxanthin. Often, lutein and zeaxanthin areadministered together.

1. Ovarian Problems

It has been found in accordance with this invention, that the use of anutraceutical, food supplement or pharmaceutical composition comprising25-OH D3, vitamin E, one or more carotenoids of this invention andascorbic acid can specifically contribute to ovarian health. Use of thenutraceutical, food supplement or pharmaceutical of this invention canlessen, reduce, ameliorate or eliminate each of these conditionsassociated with polycystic ovary syndrome.

Poultry, and in particular laying hens are a recognized experimentalmodel for human ovarian health. See, e.g. Johnson et al 2013 “The hen asa model of ovarian cancer” Nature.com/reviews/cancer 13: 432-436; andWalzem et at 2014 “Obesity-Induced Dysfunction in Female Reproduction:Lessons from Birds and Mammals”Adv. Nutr 5:199-206. Thus the datacollected in the poultry study are applicable to humans.

1. Polycystic Ovary Syndrome Symptoms:

Women with polycystic ovary syndrome often experience symptoms such asone or more of: obesity, insulin resistance, hypertriglyceridemia,increased circulating concentrations of ceramide and non-esterifiedfatty acids (NEFAs), and systemic inflammogens, including IL-1β. It hasbeen found that the combination of 25-OH D3, Vitamin C, Vitamin E and acarotenoid of this invention can ameliorate these symptoms.

Thus, another aspect of this invention is the use of the combination of25-OH D3, Vitamin C, Vitamin E and one or more carotenoids selected fromthe group consisting of: lycopene, astaxanthin, cryptoxanthin,beta-carotene, and canthaxanthin for at least one of the uses selectedfrom the group consisting of:

-   -   preventing human polycystic ovary syndrome;    -   treating human polycystic ovary syndrome;    -   delaying the onset of polycystic ovary syndrome; and    -   ameliorating a symptom of polycystic ovary syndrome.

Another aspect of this invention is a method of treating humanpolycystic ovary syndrome, ameliorating a symptom of polycystic ovarysyndrome, or delaying the onset of polycystic ovary syndrome in a personat risk of developing the syndrome, comprising:

-   -   administering to a person who has polycystic ovary syndrome or        who is at risk of developing polycystic ovary syndrome an        effective amount of a combination comprising:    -   25-hydroxyvitamin D3, Vitamin C, Vitamin E, and one or more        carotenoids selected from the group consisting of: lycopene,        astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin        and canthaxanthin. In a preferred method, the aforementioned        person is administered one or more carotenoids selected from the        group consisting of: lycopene, astaxanthin, cryptoxanthin, and        lutein.

2. Metabolic Problems

Metabolic problems associated with hyperphagia and which can belessened, reduced or eliminated through use of the feed/premix of thisinvention include:

-   -   a) clearance of non-esterified fatty acids    -   b) amelioration of plasma dyslipidemia (triglycerides,        sphingomyelin, and ceramide)    -   c) amelioration of triglyceride and ceraminde accumulation in        the liver, leg, breast muscle, and heart    -   d) suppression the tissue pro-inflammatory IL-1β production and        plasma IL-6 concentration    -   e) cardiac protection and enhanced cardiac function through the        up-regulation of the phosphorylation of STAT-3 (signal        transducer and activator of transcription 3) in the heart.    -   f) suppression of the infiltration of immune cells into the        heart    -   g) decreasing the incidence of ascites.        These above-mentioned observed improved conditions result in a        lowering of mortality rate, improved insulin signaling, reduced        lipotoxic development and systemic inflammation, and activation        of cardio-protective mechanisms against fuel-overload induced        cardiac pathogenesis.

3. Cardio-Vascular Problems

Birds in a flock can experience a “sudden death”. “Sudden Death” meansthat the individual bird died without showing previous signs or illnessor trauma. Birds appear healthy, but die rapidly with a short period ofwing beating and leg movement, during which they frequently flip ontotheir backs. They also may be found dead on their sides or breasts.There are no specific gross lesions. Recent studies indicate that deadbirds have lesions in cardiomyocytes and subendocardial Purkinje cells,and this may help in diagnosis.

We investigated this phenomenon further, as detailed in Example 3, inflocks fed ad libitum and a restricted diet, with or without thecombination of 25-OH D3 and antioxidants/anti-inflammatories. Some ofour results are set forth below.

It has been found, in accordance with this invention, that in In birdsfed ad libitum, sudden death birds which had been fed with25-OH-D3+antioxidants/anti-inflammatories had higher body weight, butlower relative liver, abdominal fat, and heart weight. In the birds withsudden death, ad libitum feeding caused cardiac adaptive hypertrophy;and some of the hypertrophic growth may develop pathologically intoventricle dilation. As a result, the heart requires a highercontractility to maintain pumping function to meet the need of bloodsupply for oxygen delivery to the peripheral tissues. This condition mayhave caused heart failure.

Importantly, we found that 25-OH-D3+antioxidants/anti-inflammatoriesdecreased cardiac pathogenic progression and thereby the incidence ofheart failure in birds fed ad libitum. Thus another aspect of thisinvention is the use of 25-OH-D3+antioxidants/anti-inflammatories toreduce the amount of cardiac problems that can lead to cardiac failure.

In both restricted and ad libitum fed birds, birds with25-OH-D3+antioxidants/anti-inflammatories exhibited less adaptivehypertrophic growth, supporting the hypothesis that most excessive fuelsmay be partitioned to the muscle, and thereby, hypertrophic growth ofthe heart for increased pumping function cannot meet the need of oxygensupply for higher growth rate (muscle) and thus may provoke cardiacarrhythmia and failure.

25-OH-D3+antioxidants/anti-inflammatories inclusion was seen to have thefollowing effects:

-   -   decreased the incidence of cardiac morbidities (dilation,        pericardial effusion, rupture) observed in necropsies of in the        dead birds.    -   Decreased the amount of irregular incidence of ECG patterns    -   Decreased arrhythmia of broiler hens fed ad libitum    -   ameliorated sudden death induced by cardiac morbidities    -   ameliorated cardiac fibrosis in hens fed ad libitum.    -   ameliorated chronic systemic inflammation in hens fed ad        libitum.    -   ameliorated cardiac cell apoptosis in hens with restricted or ad        libitum feed intake.

Thus, the 250H D3+antioxidants can protect the cardiovascular system.

Use of supplemental 25-OH D3 and antioxidants/anti-inflammatoriesameliorated deleterious effects associated has various cardiovascularbenefits. Thus this invention includes use of 25-OH D3 andantioxidants/anti-inflammatories for at least one cardiovascular benefitselected from the group consisting of:

-   -   a) clearance of non-esterified fatty acids;    -   b) amelioration of plasma dyslipidemia (triglycerides,        sphingomyelin, and ceramide);    -   c) amelioration of triglyceride and ceraminde accumulation in        the liver, leg, muscles, and heart;    -   d) suppression of the tissue pro-inflammatory IL-1β production        and plasma IL-6 concentration;    -   e) cardiac protection and enhanced cardiac function through the        up-regulation of the phosphorylation of STAT-3 (signal        transducer and activator of transcription 3) in the heart;    -   f) suppression of the infiltration of immune cells into the        heart;    -   g) decreasing the incidence of ascites;    -   h) decreasing the incidence of death due to cardiovascular        problems;    -   i) decreasing the incidence of cardiac morbidities (dilation,        pericardial effusion, rupture);    -   j) decreasing the amount of irregular incidence of ECG patterns;    -   k) decreasing the occurrence of arrhythmias;    -   l) ameliorating cardiac fibrosis;    -   m) ameliorating chronic systemic inflammation; and    -   n) ameliorating cardiac cell apoptosis    -   o) improved insulin resistance and/or blood glucose regulation.

3. Doses

In one aspect of this invention the combination of 25-OH D3 and theantioxidants/anti-inflammatories of this invention are administered tovitamin replete rather than vitamin deficient individuals. The vitaminreplete status is preferably due to the a balanced diet which suppliesat least the minimum amount of vitamins and minerals for the person. Thecombination of this invention is thus preferably used in addition to thebasic diet.

25-OH D3:

25-OH D3 is present in a daily dosage is from 1 μg to 50 μg, preferablyabout 5 μg and 25 rig. In some embodiments 10 μg is used.

Vitamin E:

The amount in a daily dosage can range from 5-750 mg/day, preferablyfrom 10-600 mg/day and more preferably from 100-500 mg per day. In someembodiments, 400 mg is used.

Ascorbic Acid:

The amount of ascorbic acid can range from 1-1000 mg/day, preferablyfrom 100-1000 mg/day. More preferably it can range from 200-1000 mg/day.

Lycopene:

The amount of lycopene can range from 1-150 mg/day; preferably 5-35mg/day.

Astaxanthin:

The amount of astaxanthin can range from 1-150 mg/day, preferably 2-50mg/day, more preferably 5-20 mg/day.

β-Cryptoxanthin

The amount of β-cryptoxanthin can range from 1-100 mg/day; preferablyfrom 2-30 mg/day; and more preferably from 5-15 mg/day.

Beta-Carotene

The amount of beta-carotene can range from 1-100 mg/day; preferably from2-20 mg/day and more preferably 5-15 mg/day.

Zeaxanthin:

The amount of zeaxanthin can range from 1-60 mg/day; preferably 2-20mg/day; and more preferably 3-10 mg/day.

Lutein: The amount of lutein can range from 1 to 100 mg/day, preferablyfrom 2-20 mg/day, and more preferably from 6-12 mg/day.

Canthaxanthin: The amount of canthaxanthin should not exceed 30 mg/day.

Representative Daily Formula 1:

-   -   25-OH D3: 5-25 μg, preferably 10 μg    -   Vitamin E 400 mg    -   Ascorbic acid: 100-1000 mg, preferably 200-1000    -   Cryptoxanthin: 0.10 mg

Representative Daily Formula #2:

-   -   25-OH D3: 5-25μg, preferably 12.5 μg    -   Vitamin E: 10 mg    -   Ascorbic acid: 100-1000 mg, preferably 200-1000    -   Lutein 6-12 mg    -   (optional) Zeaxanthin: 6 mg

Representative Daily Formula #3

-   -   25-OH D3: 5-25 μg, preferably 12.5 μg    -   Vitamin E: 200-600 mg, preferably 300-500 mg    -   Ascorbic acid: 100-1000 mg, preferably 200-1000    -   Lycopene: 20 mg

Representative Daily Formula #4

-   -   25-OH D3: 5-25 μg, preferably 10 μg    -   Vitamin E: 200-600 mg, preferably 300-500 mg    -   Ascorbic acid: 100-1000 mg, preferably 200-1000    -   Astaxanthin: 20 mg

Representative Daily Formula #5

-   -   25-OH D3: 5-25 μg, preferably 12.5 μg    -   Vitamin E: 200-600 mg, preferably 300-500 mg    -   Ascorbic acid: 100-1000 mg, preferably 200-1000    -   Beta Carotene: 10 mg

Preferred ratios include the following

Vit E Vit C Carotenoid 1-10 1-10 1 1-20 1-10 1 1-20 1-20 1 1-10 1-20 1

Optional Additional Ingredients

To each of the nutraceutical, food supplement or pharmaceuticalcomposition listed above, at least one of the additional ingredients maybe added. Preferably at least one, and more preferably more than one ofthe following ingredients are added. In other embodiments, all thefollowing ingredients are added:

-   -   Vitamin D3—The amount of vitamin D3 can range from 1-100 μg/day;        preferably from 1-50 μg/day and more preferably 5-25 μg/day.    -   Vitamin B2: The amount of Vitamin B2 can range from 0.5-300        mg/day; preferably from 5-100 mg/day and more preferably 10-50        mg/day.    -   Niacin: The amount of Niacin can range from 1-300 mg/day;        preferably from 5-100 mg/day and more preferably 10-50 mg/day.    -   Pantothenic acid: The amount of Pantothenic acid can range from        1-300 mg/day; preferably from 5-100 mg/day and more preferably        10-50 mg/day.    -   Folic acid: The amount of Folic acid can range from 50-100        μg/day; preferably from 400-800 μg/day and more preferably        400-600 μg/day.    -   Biotin: The amount of Biotin can range from 5 μg to 10 mg/day;        preferably from 30 μg to-5 mg/day and more preferably 0.1-1        mg/day.    -   Zinc: The amount of Zinc can range from 1-40 mg/day; preferably        from 5-40 mg/day and more preferably 10-20 mg/day.    -   Copper: The amount of Copper can range from 0.4-10 mg/day;        preferably from 0.7-5 mg/day and more preferably 0.9-3 mg/day.    -   Manganese: The amount of Manganese can range from 1-10 mg/day;        preferably from 1-5 mg/day and more preferably 1-3 mg/day.    -   Selenium: The amount of Selenium can range from 20-400 μg/day;        preferably from 50-200 μg/day and more preferably 50-100 μg/day.

Premixes can be made to give the above-mentioned doses and preferreddoses. One premix which forms part of this invention is formulated sothat 1 gram of premix is added to one kilogram nutraceutical, foodsupplement or pharmaceutical composition, and that the resultingnutraceutical, food supplement or pharmaceutical composition containsthe dosages described in any of the given dosages above. The amounts ofthe individual ingredients can, of course be varied so that one kilogramof premix is added to one metric ton of nutraceutical, food supplementor pharmaceutical composition and that the resulting feed contains thedosages described in any of the given dosages above. There are specificillustrations of this in the Examples, below.

Formulations

Another aspect of this invention relates to a nutraceutical compositioncomprising the combination of 25-OH D, Vitamin C, Vitamin E and at leastone carotenoid according to this invention and a nutraceuticallyacceptable carrier.

The term “nutraceutical” as used herein includes: food products,foodstuffs, dietary supplements, nutritional supplements or a supplementcomposition for a food product or a foodstuff. Thus, in anotherembodiment the present invention relates to a nutraceutical wherein thenutraceutical is a food product, foodstuff, dietary supplement,nutritional supplement or a supplement composition for a food product ora foodstuff.

As used herein, the term “food product” refers to any food or suitablefor consumption by humans. The food product may be a prepared andpackaged food (e.g., mayonnaise, salad dressing, bread, or cheese food.As used herein, the term “foodstuff” refers to any substance fit forhuman consumption. The term “dietary supplement” refers to a smallamount of a compound for supplementation of a human diet packaged insingle or multiple dose units. Dietary supplements do not generallyprovide significant amounts of calories but may contain othermicronutrients (e.g., vitamins or minerals). The term “nutritionalsupplement” refers to a composition comprising a dietary supplement incombination with a source of calories. In some embodiments, nutritionalsupplements are meal replacements or supplements (e.g., nutrient orenergy bars or nutrient beverages or concentrates).

Food products or foodstuffs are, for example, beverages such asnon-alcoholic and alcoholic drinks as well as liquid preparation to beadded to drinking water and liquid food, non-alcoholic drinks are forinstance soft drinks, sport drinks, fruit juices, such as for exampleorange juice, apple juice and grapefruit juice; lemonades, teas,near-water drinks and milk and other dairy drinks such as for exampleyoghurt drinks, and diet drinks. In another embodiment food products orfoodstuffs refer to solid or semi-solid foods comprising the compositionaccording to the invention. These forms can include, but are not limitedto baked goods such as cakes and cookies, puddings, dairy products,confections, snack foods, or frozen confections or novelties (e.g., icecream, milk shakes), prepared frozen meals, candy, snack products (e.g.,chips), liquid food such as soups, spreads, sauces, salad dressings,prepared meat products, cheese, yogurt and any other fat or oilcontaining foods, and food ingredients (e.g., wheat flour). The term“food products” or “foodstuffs” also includes functional foods andprepared food products, the latter referring to any pre-packaged foodapproved for human consumption.

Dietary supplements of the present invention may be delivered in anysuitable format. In preferred embodiments, dietary supplements areformulated for oral delivery. The ingredients of the dietary supplementof this invention are contained in acceptable excipients and/or carriersfor oral consumption. The actual form of the carrier, and thus, thedietary supplement itself, is not critical. The carrier may be a liquid,gel, gelcap, capsule, powder, solid tablet (coated or non-coated), tea,or the like.

The dietary supplement is preferably in the form of a tablet or capsule.Suitable excipient and/or carriers include maltodextrin, calciumcarbonate, dicalcium phosphate, tricalcium phosphate, microcrystallinecellulose, dextrose, rice flour, magnesium stearate, stearic acid,croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose,vegetable gums, lactose, methylcellulose, povidone,carboxymethylcellulose, corn starch, and the like (including mixturesthereof). Preferred carriers include calcium carbonate, magnesiumstearate, maltodextrin, and mixtures thereof. The various ingredientsand the excipient and/or carrier are mixed and formed into the desiredform using conventional techniques. The tablet or capsule of the presentinvention may be coated with an enteric coating that dissolves at a pHof about 6.0 to 7.0. A suitable enteric coating that dissolves in thesmall intestine but not in the stomach is cellulose acetate phthalate.Further details on techniques for formulation for and administration maybe found in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

In other embodiments, the dietary supplement is provided as a powder orliquid suitable for adding by the consumer to a food or beverage. Forexample, in some embodiments, the dietary supplement can be administeredto an individual in the form of a powder, for instance to be used bymixing into a beverage, or by stirring into a semi-solid food such as apudding, topping, sauce, puree, cooked cereal, or salad dressing, forinstance, or by otherwise adding to a food e.g. enclosed in caps of foodor beverage container for release immediately before consumption. Thedietary supplement may comprise one or more inert ingredients,especially if it is desirable to limit the number of calories added tothe diet by the dietary supplement. For example, the dietary supplementof the present invention may also contain optional ingredientsincluding, for example, herbs, enhancers, colorants, sweeteners,flavorings, inert ingredients, and the like.

The following non-limiting Examples are presented to better illustratethe invention.

EXAMPLES Example 1

A total of thirty 45-week-old broiler breeder hens (ROSS 308) wereobtained from a commercial flock for the study. A basal broiler breederlaying diet was formulated as shown in Table 1. The calculated nutrientcomposition is shown in Table 2.

TABLE 1 Ingredient composition of the basal broiler breeder layingdiets. Composition %, w/w Corn 66.9 Soybean meal 22.2 Oil fat 1.67 CaCarbonate 6.36 (ground oyster shell) Dicalcium phosphate 1.8 Choline-Cl(70%) 0.1 Mineral Premix¹ 0.1 Copper sulfate 0.05 Vitamin Premix² 0.1¹Mineral premix provided (per kg of diet for treatment groups 1, 2 and3): Cu 18 mg; | 1.1 mg; Fe 80 mg; Mn 150 mg; Zn 125 mg; and Se 0.25 mg.²Refer to Table 2 for further detail.

TABLE 2 Vitamin premix composition (provided per kg of diet) Treatment 33 = ad libitum Treatments 1 and 2 feeding + 25-OH-D3 + 1 = restrictedfeeding antioxidants/anti- Vitamin 2 = ad libitum feeding inflammatoriesA (IU) 10000 12000 D3 (IU) 2500 3000 E (mg) 100 150 K3 (mg) 3 5 B1 (mg)3 5 B2 (mg) 8 14 B6 (mg) 6 8 B12 (mg) 0.03 0.03 Niacin (mg) 60 120Pantothenic acid (mg) 18 30 Folic acid (mg) 1 4 Biotin (mg) 0.2 0.4 C(ascorbic acid) (mg) 0 150 25-OH-D3 (mcg) 0 69 Canthaxanthin (mg) 0 6

TABLE 3 Calculated nutrient composition (%) of the basal broiler breederlaying diets. Composition % w/w Crude protein 16 Crude fat 4.2 Calcium3.1 Sodium 0.16 Total Phosphorus 0.64 Total ME 2910 kcal/kg

Diet was supplemented with or without 25-OH D3 at 69 mcg/kg diet incombination with antioxidants/anti-inflammatories (vitamin E, ascorbicacid, canthaxanthin) and enriched levels of selected vitamins. Hens wererandomly allocated to 3 treatment groups according to feeding regimen(restricted and ad libitum) as follows:

Basal diet—restricted feeding (140 g/day)

Basal diet—ad libitum feeding

Basal diet—ad libitum feeding+25-OH-D3 at 69 mcg/kgdiet+antioxidants/anti-inflammatories

They were individually-housed in wire cages placed in a controlled roomwith 14 h:10 h light:dark period and at a temperature of 25±3° C. Waterwas available ad libitum. The experimental period was lasted for 10weeks. Three weeks after the feeding trial, some birds were used forrelevant plasma parameter analyses. At the end of experiment, hens wereeuthanized and sacrificed for tissue sample collection for furtherstudies: Necropsy of tissue morphology

-   -   Determination of lipid and sphingolipid profile—serum and        tissues    -   Determination of tissue pro-inflammatory cytokines    -   Determination of insulin resistance    -   Collection of tibia for bone strength analysis    -   Harvesting heart (cardiomyopathy) and skeletal muscle (breast        and thigh) for    -   myopathy analyses.

Example 2 Results and Discussion

25-OH D3 and Antioxidants/Anti-Inflammatories Suppressed Adiposity andAbdominal Fat in Overfed Broiler Hens

Breeder hens are capable of storing large quantities of excess energy(in the form of triglycerides) in the liver, adipose tissue and yolk ofdeveloping oocytes. Lipogenesis (i.e., the conversion of glucose totriglycerides) takes place primarily in the liver of birds and involvesa series of linked, enzyme catalyzed reactions including glycolysis, thecitric acid cycle and fatty acid synthesis. Hepatic lipogenesis issubject to both nutritional and hormonal control and is highlyresponsive to changes in the diet. Adipose tissue serves primarily as astorage site for lipid with little lipogenic activity. Differentiallipogenic capacity of liver vs. adipose tissue in birds is a function ofthe expression of a key transcription factor, sterol regulatory elementbinding protein-1 (SREBP-1). The gene for SREBP-1 is highly expressed inthe liver, but to a much lesser extent in adipose tissue. Moreover, theexpression of a number of lipogenic enzyme genes such as fatty acidsynthase, malic enzyme, acetyl CoA carboxylase, ATP citrate lyase andsteroyl CoA desaturase 1 is directly influenced by SREBP-1.

Breeder hens fed ad libitum accreted more abdominal fat than thoserestricted fed. Dietary supplementation of 25-OHD3 andantioxidants/anti-inflammatories ameliorated the deleterious effect ofad libitum feeding on body and tissue weight, particularly on relativeadipose tissue weight (adiposity) (Table 4). Tibial strength of adlibitum-fed hens was enhanced by 25-OH D3 andantioxidants/anti-inflammatories.

TABLE 4 25-OH D3 and antioxidants/anti-inflammatories on body weight,liver weight, abdominal fat weight and tibial strength of ad libitum-fedbroiler breeder hens Ad libitum Ad feeding + 25-OH-D3 + Restrictedlibitum Antioxidants + Pooled feeding feeding anti-inflammatories SEM¹Body weight (kg) 3.67^(c) 4.60^(a) 4.04^(b) 0.26 Liver weight (g)41.4^(b) 48.5^(a)  49.6^(a) 5.6 Liver/body weight (%) 1.14 1.06  1.230.18 Abdominal fat weight (g) 40.9^(c) 185.9^(a)   110.8^(b) 28.9Abdominal fat weight/ 1.10^(c) 4.01^(a) 2.74^(b) 0.70 body weight (%)Tibial strength (kg/cm2) 32.3^(b) 38.3^(ab )  44.3^(a) 3.57 ^(a-c)Withina row, means without a common superscript differ (P < 0.05). ¹Pooledstandard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Lowered Mortality andImproved Egg Production, Ovarian Morphology and Plasma 176 EstradiolLevel

Secretion of estradiol is the hallmark of successful ovulatoryfollicles. In addition to its role in triggering the preovulatory surgeof gonadotropins, estradiol is an important intra-ovarian growth,differentiation, and survival factor. Inclusion of 25-OH D3 andantioxidants/anti-inflammatories reduced mortality and incidence ofovarian degeneration and ovarian-tumor-like morphology, increased eggproduction and sustained plasma estradiol levels in birds under adlibitum feed intake.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated ImpairedGlucose Clearance and Insulin Sensitivity

Dietary inclusion of 25-OH D3 and antioxidants/anti-inflammatoriesimproves insulin resistance as evidenced by ameliorating fasting plasmaglucose and non-esterifed fatty acid level in overfed hens for 10 weeks(Table 6). In glucose clearance test, lean hens showed a very sharpclearance rate between 30-60 min after glucose infusion, and converselyobese hens had a very sluggish clearance rate between 30-90 min (FIG.1). In insulin secretion, obese hens showed a higher plasma insulinlevel under fasting status and after glucose infusion when compared tolean hens (FIG. 1). Both glucose clearance and glucose-induced insulinsecretion were corrected by 25-OH D3 andantioxidants/anti-inflammatories inclusion in overfed hens for 3 weeks(FIG. 1).

TABLE 5 25-OH D3 and antioxidants/anti-inflammatories on plasma glucose,non-esterified fatty acid (NEFA) and insulin of ad libitum-fed broilerbreeder hens Ad libitum Ad feeding + 25-OH-D3 ++ Restricted libitumantioxidants/ Pooled feeding feeding anti-inflammatories SEM¹ After 3weeks of feeding Plasma glucose (mg/dL) 181.5 202.5 188.5 11.9 PlasmaNEFA (μmole/mL) 0.21^(b) 0.35^(a) 0.25^(b) 0.05 After 10 weeks offeeding Plasma glucose (mg/dL) 180.6^(b) 212.6^(a) 195.6^(b) 12.7 PlasmaNEFA (μmole/mL) 0.35^(b) 0.44^(a) 0.33^(b) 0.05 Fasting plasma insulin1.38 1.15 1.59 0.21 Glucose-induced insulin 2.66a 1.97b 2.46a 0.36^(a-b)Within a row, means without a common superscript differ (P <0.05). ¹Pooled standard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated Dyslipidemia

Ad libitum-fed hens elevated plasma triglyceride, ceramide andsphingomyelin levels. However, supplementation of combined 25-OH D3 andantioxidants/anti-inflammatories lowered the level of these lipidmetabolites in the plasma of ad libitum-fed hens (Table 6).

TABLE 6 25-OH D3 and antioxidants/anti-inflammatories on plasmatriacyglycerol, ceramide and sphingomyelin of ad libitum-fed broilerbreeder hens Ad libitum Ad feeding + 25-OH-D3 ++ Restricted libitumantioxidants/ Pooled feeding feeding anti-inflammatories SEM¹ After 3weeks of feeding Plasma triacyglycerol (mg/mL) 15.6 17.4 14.5 2.9 Plasmaceramide (nmole/mL) 11.5^(b) 18.2^(a) 13.5^(b) 2.83 Plasma sphingomyelin(μmole/mL) 0.14^(b) 0.28^(a) 0.16^(b) 0.05 After 10 weeks of feedingPlasma triacyglycerol (mg/mL) 12.75^(b) 15.2^(a) 11.5^(b) 2.2 Plasmaceramide (nmole/mL) 8.1^(b) 12.3^(a) 8.8^(b) 1.65 Plasma sphingomyelin(μmole/mL) 0.15^(b) 0.22^(a) 0.12^(b) 0.05 ^(a-b)Within a row, meanswithout a common superscript differ (P < 0.05). ¹Pooled standard errorof the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Reduced Accumulation ofTissue Triglyceride and Ceramide Content

Accumulation of triglyceride and ceramide in the liver, heart and legmuscles was lower in hens fed supplemental 25-OH D3 andantioxidants/anti-inflammatories than in those fed ad libitum (Table 7).

TABLE 7 25-OH D3 and antioxidants/anti-inflammatories on tissuetriacyglycerol and ceramide content of ad libitum-fed broiler breederhens Ad libitum Ad feeding + 25-OH-D3 + Restricted libitumAntioxidants + Pooled feeding feeding anti-inflammatories SEM¹Triacyglycerol (mg/g tissue) Liver 69.0^(b) 94.8^(a) 79.8^(a) 10.6 Heart33.1^(b) 55.6^(a) 45.6^(a) 6.8 Breast muscle 15.6 18.0 17.4 0.05 Legmuscle 30.2^(c) 52.3^(a) 41.2^(b) 6.6 Ceramide (mg/g tissue) Liver174.5^(b) 287.9^(a) 235.0^(a) 52.7 Heart 17.5^(c) 30.2^(a) 23.5^(b) 2.4Breast muscle 2.25 2.91 2.52 0.54 Leg muscle 4.12^(b) 7.12^(a) 6.01^(a)0.85 ^(a-b)Within a row, means without a common superscript differ (P <0.05). ¹Pooled standard error of the mean.

25-OH D3 and antioxidants/anti-inflammatories depressed tissueproinflammatory IL-1β Production and Plasma IL-6 Concentrations inOverfed Broiler Hens

Obesity-associated inflammation was ameliorated by dietary 25-OH D3 andantioxidants/anti-inflammatories supplementation as evidenced bysuppressed circulating IL-6 levels and IL-1β production in adiposetissue, liver, leg and breast muscle, and heart (FIG. 2).

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated Lipotoxicityin Broiler Breeder Hens Fed Ad Libitum

A central complication of obesity is the development of insulinresistance, which is when insulin is incapable of eliciting postprandialnutrient storage in its primary target tissues, skeletal muscle andliver. Without wishing to be bound by theory, it appears that twoprobable mechanisms may explain how increased adipose stores affectoverall insulin sensitivity throughout the body, contributing to thedown regulation of insulin signaling in peripheral tissues. Firstly, thedelivery of nutrients to cells or tissues is in excess of their storagecapacities and thus this leads to the generation of metabolites thatinhibit insulin action. Of particular importance, lipid derivatives,such as triacylglycerol and ceramide, have been shown to inhibitspecific insulin signaling intermediates, thus blocking postprandialglucose uptake and/or glycogen synthesis. In the case of broiler breederfemales being fed ad libitum, the persistent accumulation of thesemetabolites in peripheral tissues likely contributes to a sustainedstate of insulin resistance throughout the hen and of lipotoxicdevelopment. Secondly, increased adiposity induces a chronicinflammatory state characterized by elevated circulating levels ofpro-inflammatory cytokines produced from adipocytes or from macrophagesinfiltrating the fat pad. These inflammatory mediators have been shownto antagonize insulin signaling directly, and also to induce catabolicprocesses, thus further increasing the delivery of nutrient metabolitesto insulin-responsive organs.

Overall, excess supply of glucose leading to the formation of excesssaturated fatty acids and therefore accumulation of lipids innon-adipose tissues elevates the cellular levels of -active lipids(sphingolipids) that inhibit the signaling pathways implicated inmetabolic regulation together with activated inflammatory responses andlipotoxic development. In particular, ceramide is a putativeintermediate linking both excess nutrients (i.e., saturated fatty acids)and inflammatory cytokines to the induction of insulin resistance.Moreover, ceramide is toxic in a variety of different cell types and iscapable of damaging the heart, pancreas and vasculature. Moreover,25-hydroxy D3 and antioxidants/anti-inflammatories were effective inameliorating the deleterious effect of metabolic and endocrinedysregulations and pro-inflammatory responses resulting from increasedadiposity occurring in broiler breeder hens fed to satiation.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorate CardiacMorbidities, Ascites, and Inflammation in Overfed Broiler Hens

The heart may become dysfunctional due to excess lipid accumulation.That ad libitum feeding promoted triglyceride accumulation in the heartsuggested that increased cardiac fatty acid availability is adaptivelyesterified into triglyceride. In addition, ceramide content of the heartwas also increased as a result of ad libitum feeding. Ceramide is acardiotoxin in lipotoxic cardiomyopathy, which elicited inflammatoryresponses as evidenced by more cardiac infiltration of immune cells.(Table 9).

TABLE 8 25-OH D3 and antioxidants/anti-inflammatories on cardiacresponses of ad libitum-fed broiler breeder hens Ad libitum Ad feeding +25-OH-D3 + Restricted libitum antioxidants + Pooled feeding feedinganti-inflammatories SEM¹ Heart weight (g) 14.5^(b) 19.2^(a) 17.3^(a) 1.8Heart/body weight (%) 0.40 0.47 0.43 0.17 Heart septum (HS) weight (g)2.73 3.01 2.83 0.57 HS weight/heart weight (%) 18.7 14.6 15.7 3.9 Rightatrium (RA) wall weight (g) 1.20^(b) 1.97^(a) 1.72^(ab) 0.38 RA wallweight/heart weight (%) 8.2 9.6 9.6 1.9 Right ventricle (RV) wall weight(g) 0.95^(b) 1.51^(a) 1.73^(a) 0.27 RV wall weight/heart weight (%)6.3^(b) 7.5^(ab) 9.4^(a) 1.5 Left atrium (LA) wall weight (g) 1.17^(b)2.26^(a) 2.02^(a) 0.43 LA wall weight/heart weight (%) 12.2 11.1 10.93.1 Left ventricle (LV) wall weight (g) 3.78^(b) 4.45^(a) 4.65^(a) 0.34LV wall weight/heart weight (%) 25.5^(a) 21.7^(b) 25.8^(ab) 2.4Incidence of transudate within 1/7 5/10 3/10 pericardium (heart/total)Incidence of heart ventricle dilation 1/7 6/10 3/10 (heart/total)Incidence of ascites (hen/total) 0/7 3/10 1/10 Cardiac immune cell count97.9^(a) 127.7^(a) 57.7^(b) 32.4 (cells/mm2) ^(a-b)Within a row, meanswithout a common superscript differ (P < 0.05). ¹Pooled standard errorof the mean.

Cardiac hypertrophy represents clinically an adaptive response toincreased workload on the heart. However, cardiac responses to neuraland hormonal factors can also incite hypertrophic changes independent ofincreases in afterload or vascular resistance. Fuel overloading-inducedcardiac compensatory growth occurred in broiler breeder hens

(Table 8). Cardiac hypertrophy may become maladaptive and eventuallydevelop into pathological conditions, leading to heart failure. Theseresults supported the fact that lipotoxic development and hypertrophicgrowth in the heart tend to elicit inflammatory responses.

The cardioprotective role of phosphorylated STAT-3 (signal transducerand activator of transcription 3) is becoming increasingly clear inrecent years. Interestingly, combined 25-OH D3 andantioxidants/anti-inflammatories induced greater activation of STAT-3(i.e., phosphorylation of STAT-3) in the heart than restricted-fedbreeder hens (FIG. 3), with the lowest activation being observed in adlibitum-fed broiler breeder hens. The incidence of transudate fluidwithin pericardium, heart ventricle dilation and ascites was alleviatedin ad-libitum-fed breeder hens when supplemented with combined25-hydroxy D3 and antioxidants/anti-inflammatories.

Example 3 Cardio-Myopathy Trial

Materials and Methods

A total of thirty 45-week-old broiler breeder hens (ROSS 308) wereobtained from a commercial flock for the study. A basal broiler breederlaying diet was formulated as shown in Table 12. The calculated nutrientcomposition is shown in Table 13.

TABLE 12 Ingredient composition of the basal broiler breeder layingdiets. Composition %, w/w Corn 66.9 Soybean meal 22.2 Oil fat 1.67Calcium carbonate 6.36 (ground oyster shell) Dicalcium phosphate 1.8Salt 0.08 Choline-Cl (70%) 0.1 Mineral premix¹ 0.1 Cooper sulfate 0.05Vitamin premix² 0.1 ¹Mineral premix provided (per kg of diet fortreatment groups 1, 2 and 3): Cu, 18 mg; I, 1.1 mg; Fe, 80 mg; Mn, 150mg; Zn, 125 mg; and Se, 0.25 mg. ²Refer to Table 13, below for furtherdetail.

TABLE 13 Vitamin premix composition (provided per kg of diet) Treatments2 and 4 2 = restricted feeding + 25-OH-D3 + antioxidants/anti-inflammatories Treatments 1 and 3 4 = ad libitum feeding + 1 =restricted feeding 25-OH-D3 + antioxidants/ Vitamin 3 = ad libitumfeeding anti-inflammatories A (IU) 10000 12000 D3 (IU) 2500 3000 E (mg)100 150 K3 (mg) 3 5 B1 (mg) 3 5 B2 (mg) 8 14 B6 (mg) 6 8 B12 (mg) 0.030.03 Niacin (mg) 60 120 Pantothenic acid 18 30 (mg) Folic acid (mg) 1 4Biotin (mg) 0.2 0.4 C (mg) 0 150 25-OH-D3 (mcg) 0 69 Canthaxanthin (mg)0 6

TABLE 14 Calculated nutrient composition (%) of the basal broilerbreeder laying diets. Composition %, w/w Crude protein 16 Crude fat 4.2Calcium 3.1 Potassium 0.44 Sodium 0.16 Total phosphorus 0.64 Total ME2910 kcal/kg

Diet was supplemented with or without Hy⋅D® at 69 mcg 25-OH-D3/kg dietin combination with antioxidants (ascorbic acid, canthaxanthin) andenriched levels of selected vitamins. Hens were randomly allocated totreatment groups according to feeding regimen (restricted and adlibitum) as follows:

-   -   1. Basal diet—restricted feeding (140 g/day)    -   2. Basal diet—restricted feeding+Hy⋅D® (25-OH-D3 at 69 mcg/kg        diet)+antioxidants/anti-inflammatories    -   3. Basal diet—ad libitum feeding    -   4. Basal diet—ad libitum feeding+Hy⋅D® (25-OH-D3 at 69 mcg/kg        diet)+antioxidants/anti-inflammatories

Results:

TABLE 15 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti-inflammatories on egg production of broiler hens withrestricted or ad libitum feed intake. Restriction + 25- Ad libitum +OH-D3 + 25-OH-D3 + antioxidant/ antioxidant/ anti- anti- Restrictioninflammatories Ad libitum inflammatories Dead Dead Dead Dead Whole birdWhole bird Whole bird Whole bird flock flock flock flock flock flockflock flock (n = 68) (n = 19) (n = 70) (n = 11) (n = 80) (n = 58) (n =79) (n = 47) PROD  51.4 ± 2.5^(a) 47.2 ± 1.9^(a)  51.3 ± 2.7^(a) 40.2 ±1.8^(b) 32.0 ± 2.9^(b) 43.4 ± 1.7^(ab) 34.8 ± 2.7^(b) 45.8 ± 2.1^(ab)YIELD 129.0 ± 5.5^(b) 47.9 ± 2.5^(b) 143.2 ± 5.7^(a) 33.3 ± 1.9^(b) 41.1± 5.3^(d) 41.5 ± 2.1^(b ) 54.7 ± 5.9^(c) 41.1 ± 2.0^(b ) PROD = Eggproduction rate (eggs/day/hen, %); YIELD = Egg yield (eggs/hen)

Results were expressed with mean±SEM.

Means with different superscript letters are significantly differentwithin the same flock (P<0.05).

FIG. 7 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on egg production of broilerhens with restricted or ad libitum feed intake. From TABLE 15 and FIG.7, it can be concluded that:

-   1. 25-OH-D3+antioxidants/anti-inflammatories improved total egg    yield by promoting survival.-   2. In dead birds, 25-OH-D3+antioxidants/anti-inflammatories had no    effect on egg yield and egg production rate in bird fed ad libitum,    but decreased egg yield and egg production rate in restricted birds.    In the whole flock, however,    25-OH-D3+antioxidants/anti-inflammatories increased egg yield but    not egg production rate.-   3. These results suggested that    25-OH-D3+antioxidants/anti-inflammatories accelerated the    progression into death in restricted birds that are susceptible to    sudden death, and thus acting as a flock culler to exclude the    sudden death-susceptible birds for longer survival and thereby    reduced the flock maintenance cost.

TABLE 16 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti-inflammatories on mortality of broiler hens withrestricted or ad libitum feed intake and body characteristics of thedead hens Restriction + Ad libitum ++ 25-OH-D3 + 25-OH-D3 + antioxidant/antioxidant/ Restriction anti-inflammatories Ad libitumanti-inflammatories (n = 19) (n = 11) (n = 58) (n = 47) Mortality 19/6811/70 58/80 47/79 (dead birds (26.47%) (15.71%) (72.5%) (59.49%) of thetotal) Body weight 3.91 ± 0.090^(C)     4.01 ± 0.084^(bc) 4.07 ±0.083^(b)    4.37 ± 0.082^(a) of the dead birds (kg) Liver weight 89.10± 6.79^(b)    99.27 ± 7.92^(ab) 105.52 ± 5.48^(a)   106.70 ± 4.68^(a) ofthe dead birds (g) Relative 2.28 ± 0.0016^(d)   2.47 ± 0.0020^(b) 2.59 ±0.0011^(a)   2.44 ± 0.0009^(c) liver weight of the dead birds (g/100 gBW) Abdominal 60.45 ± 4.56^(b)   68.54 ± 6.05^(b) 148.64 ± 8.45^(c)  133.54 ± 9.44^(a) fat weight of the dead birds (g) Relative 1.55 ±0.0013^(d)    1.71 ± 0.0016^(c) 3.65 ± 0.0014^(a)   3.06 ± 0.0009^(b)abdominal fat weight of the dead birds (g/100 g BW) Heart 18.12 ±0.81^(c)    19.93 ± 0.87^(b) 23.66 ± 0.96^(a)    22.29 ± 0.86^(a) weightof the dead birds (g) Relative  0.46 ± 0.00023^(c)   0.50 ± 0.00021^(b) 0.58 ± 0.00024^(a)     0.51 ± 0.00018^(b) heart weight of the deadbirds (g/100 g BW)

Results were expressed with mean±SEM.

Means with different superscript letters are significantly different(P<0.05)

FIG. 8 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on the survival rate ofbroiler hens with restricted or ad libitum feed intake

From Table 16 and FIG. 8 it can be concluded that:

-   1. 25-OH-D3+antioxidants/anti-inflammatories improved bird survival    rate.-   2. In birds fed ad libitum, dead birds with    25-OH-D3+antioxidants/anti-inflammatories had higher body weight,    but lower relative liver, abdominal fat, and heart weight,    suggesting that most excessive fuels may be partitioned to the    muscle, and thereby, adaptive hypertrophic growth of the heart for    increased pumping function cannot meet the need of oxygen supply for    higher growth rate (muscle) and thus may provoke cardiac arrhythmia    and failure.

TABLE 17 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti- inflammatories on carcass characteristics at age of35 and 50 weeks of broiler hens with restricted or ad libitum feedintake Restriction + Ad libitum + 25-OH-D3 + 25-OH-D3 +antioxidant/anti- antioxidant/anti- Restriction inflammatories Adlibitum inflammatories (n = 6) (n = 6) (n = 6) (n = 6) Body wt (kg): at35 wks 3.57 ± 0.19^(b) 3.66 ± 0.15^(b) 4.32 ± 0.27^(a) 4.36 ± 0.33^(a)at 50 wks 3.78 ± 0.21^(b) 3.83 ± 0.23^(b) 4.62 ± 0.34^(a) 4.71 ±0.38^(a) Liver wt (g): at 35 wks 55.35 ± 4 42^(b)  57.37 ± 2 27^(b) 98.45 ± 4.76^(a)  83.37 ± 4.17^(b)  at 50 wks 58.67 ± 3.84^(b)  59.75 ±4.67^(b)  72.56 ± 5.14^(a)* 68.45 ± 3.42^(b)* Relative liver wt (g/100 gBW, %): at 35 wks 1.55 ± 0.06^(d) 1.57 ± 0.11^(b) 2.28 ± 0.10^(a) 1.91 ±0.09^(b) at 50 wks 1.55 ± 0.05  1.56 ± 0.14   1.57 ± 0.13*  1.45 ± 0.07*Abdominal fat wt of the dead birds (g): at 35 wks 47.63 ± 2.16^(c) 50.57 ± 3.64^(c)  123.84 ± 9.84^(a)  95.47 ± 8.12^(b)  at 50 wks 51.24 ±2.38^(c)  53.12 ± 2.57^(c)  147.62 ± 8.55^(c)*  112.47 ± 9.01^(c) Relative abdominal fat wt (g/100 g BW, %): at 35 wks 1.35 ± 0.11^(c)1.38 ± 0.06^(c) 2.86 ± 0.0^(a)  2.19 ± 0.03^(b) at 50 wks 1.36 ±0.12^(c) 1.39 ± 0.50^(c)  3.20 ± 0.06^(a)*  2.39 ± 0.08^(b)* Heart wt(g): at 35 wks 12.69 ± 0.38^(b)  12.09 ± 0.31^(b)  17.33 ± 0.65^(a) 17.76 ± 0.70^(a)  at 50 wks 14.12 ± 0.41^(b)   13.88 ± 0.0.36^(b) 22.21± 0.71^(a)* 20.88 ± 0.73^(a)* Relative heart wt (g/100 g BW, %): at 35wks 0.357 ± 0 024^(b) 0.331 ± 0.011^(b) 0.401 ± 0 016^(a)  0.414 ±0.046^(ab) at 50 wks 0.374 ± 0.025^(b) 0.362 ± 0.024^(b)  0.481 ±0.015^(a)*  0.443 ± 0.024^(ab) Results were expressed with mean ± SEM.Means with different superscript letters are significantly different (P< 0.05) *significant difference vs. age at 35 wks.

The low relative liver weight in ad libitum birds at age of 50 weeksappears to be due to ovarian regression developed and thus decreasedestrogen secretion leading to decreased lipid synthesis in the liver foryolk deposition.

FIG. 9 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on body weight of broiler henswith restricted or ad libitum feed intake.

TABLE 18 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti-inflammatories on the cardiac morbidities of the deadhens with restricted or ad libitum feed intake Restriction + Adlibitum + 25-OH-D3 + (25-OH-D3 + antioxidant/anti- antioxidant/anti-Restriction inflammatories Ad libitum inflammatories (n = 19) (n = 11)(n = 58) (n = 47) Mortality 19/68 11/70 58/80 47/79  (dead birds(26.47%) (15.71%) (72.5%) (59.49%) of the total) Cardiac morbidities(birds of the death) 1. hypertrophy 6/19 0/11 20/58 14/47  2. ventricle3/19 3/11 16/58 10/47  dilation 3. effusion in 6/19 0/11 26/58 15/47 the pericardial cavity 4. ascites 2/19 0/11  7/58 5/47 5. myocardial1/19 0/11  6/58 4/47 rupture trauma 1 + 3 2/19 0/11 10/58 7/47 2 + 32/19 1/11 11/58 6/47 1 + 4 2/19 0/11  3/58 0/47 3 + 4 1/19 0/11  2/581/47 2 + 5 0/19 0/11  3/58 1/47 2 + 3 + 4 0/19 0/11  2/58 1/47

Results were expressed as a ratio.

FIGS. 10-11 illustrate the gross morphology of the heart of dead henswith dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitumfeed intake.

Conclusions and annotations from Tables 17 and 18 and FIGS. 9-11:

-   1. Ad libitum feeding caused cardiac adaptive hypertrophy, and some    of the hypertrophic growth may develop pathologically into ventricle    dilation. As a result, the heart requires a higher contractility to    maintain pumping function to meet the need of blood supply for    oxygen delivery to the peripheral tissues and thus may provoke heart    failure.-   2. 25-OH-D3+antioxidants/anti-inflammatories decreased the incidence    of cardiac morbidities (dilation, pericardial effusion, rupture) in    the dead birds.-   3. In both restricted and ad libitum fed birds, birds with    25-OH-D3+antioxidants/anti-inflammatories exhibited less adaptive    hypertrophic growth. This supports the hypothesis that most    excessive fuels may be partitioned to the muscle, and thereby,    hypertrophic growth of the heart for increased pumping function    cannot meet the need of oxygen supply for higher growth rate    (muscle) and thus may provoke cardiac arrhythmia and failure.

TABLE 19 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti-inflammatories on electrocardiogram (ECG) pattern andarrhythmia of broiler hens with restricted or ad libitum feed intake.See FIGS. 12 and 13 for examples of the EGC patterns. Restriction + Adlibitum + 25-OH-D3 + 25-OH-D3 + antioxidant/anti- antioxidant/anti-Restriction inflammatories Ad libitum inflammatories (n = 8) (n = 8) (n= 8) (n = 8) ECG pattern A at 4/8 4/8 1/8 3/8 age of 35 wks ECG patternB or C 2/8 3/8 2/8 3/8 at age of 35 wks ECG pattern D, E, F, 2/8 1/8 5/82/8 or G at age of 35 weeks ECG pattern A at 3/8 4/8 0/8 2/8 age of 50wks ECG pattern B or C 3/8 2/8 1/8 3/8 at age of 50 wks ECG pattern D,E, F, 2/8 2/8 7/8 3/8 or G at age of 50 weeks Arrhythmic ECG 0/8 0/8 2/81/8 pattern at age of 35 wks Arrhythmic ECG 1/8 1/8 4/8 2/8 pattern atage of 50 wks

Conclusions and annotations from Table 19 and FIGS. 12 and 13:

-   -   25-OH-D3+antioxidants/anti-inflammatories decreased irregular        incidence of ECG pattern (pattern D to G) and arrhythmia of        broiler hens fed ad libitum and ameliorated sudden death induced        by cardiac morbidities.

FIG. 14 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on cardiac fibrosis of broilerhens with restricted or ad libitum feed intake. (at age of 35 weeks)Means with letters over the bars are significantly different (P<0.05)

Conclusions and annotations from FIG. 14:

-   1. 25-OH-D3+antioxidants/anti-inflammatories ameliorated cardiac    fibrosis in hens fed ad libitum.

FIG. 15 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on plasma IL-6 (top graphs)and IL-1β concentration (lower graphs) of broiler hens with restrictedor ad libitum feed intake. Results were expressed with mean±SEM (n=6).Means with different letters over the bars are significantly different(P<0.05)

Conclusions and annotations from FIG. 15:

-   -   25-OH-D3+antioxidants/anti-inflammatories ameliorated chronic        systemic inflammation in hens fed ad libitum.

FIG. 16 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on cardiac cell apoptosis ofbroiler hens with restricted or ad libitum feed intake. (at age of 35weeks). Results were expressed with mean±SEM (n=3). Means with differentletters over the bars are significantly different (P<0.05)

Conclusions and annotations from FIG. 16:

-   1. 25-OH-D3+antioxidants/anti-inflammatories ameliorated cardiac    cell apoptosis in hens with restricted or ad libitum feed intake.

CONCLUSIONS

Supplemental 25-OH D3 and antioxidants/anti-inflammatories ameliorateddeleterious effects associated with overfeeding of broiler breederfemales by:

-   -   lowering mortality rate and improving ovary function and        therefore reproductive performance of overfed broiler breeder        hens.    -   improving endocrine (insulin) signaling    -   reducing lipotoxic development and systemic inflammation    -   activating cardio-protective mechanisms against fuel-overload        induced cardiac pathogenesis.

1. A composition, nutraceutical, or pharmaceutical composition suitablefor human use comprising: 25-Hydroxy vitamin D (25-OH D), Vitamin C,Vitamin E and one or more carotenoids selected from the group consistingof: lycopene, astaxanthin, cryptoxanthin, beta-carotene, lutein,zeaxanthin and canthaxanthin.
 2. A composition, nutraceutical, orpharmaceutical composition according to claim 1 further comprising atleast one bio-active ingredient selected from the group consisting of:Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid,Biotin, Zinc, Copper, Manganese, and Selenium.
 3. A composition,nutraceutical or pharmaceutical composition selected from the groupconsisting of: Formula 1: 25-OH D: 5-25 μg, Vitamin E: 200-600 mg,Ascorbic acid: 100-1000 mg and Cryptoxanthin: 0.10 mg; Formula 2: 25-OHD: 5-25 μg; Vitamin E: 200-600 mg, Ascorbic acid: 100-1000 mg, Lutein6-12 mg; and optionally Zeaxanthin: 6 mg; Formula 3: 25-OH D: 5-25 μg;Vitamin E: 200-600 mg, Ascorbic acid: 100-1000 mg, and Lycopene: 20 mg;Formula 4: 25-OH D: 5-25 m; Vitamin E: 200-600 mg, Ascorbic acid:100-1000 mg, and Astaxanthin: 20 mg; Formula #5: 25-OH D: 5-25 μg,Vitamin E: 200-600 mg, Ascorbic acid: 100-1000 mg, and Beta Carotene: 10mg
 4. A composition, nutraceutical, pharmaceutical composition, orpremix according to claim 1 wherein the 25-hydroxy vitamin D is25-hydroxy vitamin D3 (25-OH D3).
 5. A composition, nutraceutical,pharmaceutical composition, or premix according to claim 1 for the usein treating, preventing, delaying the onset or ameliorating a symptom ofhuman polycystic ovarian syndrome.
 6. A composition, nutraceutical,pharmaceutical composition, or premix according to claim 1 for the usein ameliorating weight gain in a person who is hyperphagic.
 7. Acomposition, nutraceutical, pharmaceutical composition or premixaccording to claim 1 for the use in conferring at least onecardiovascular benefit selected from the group consisting of: a)clearance of non-esterified fatty acids; b) amelioration of plasmadyslipidemia (triglycerides, sphingomyelin, and ceramide); c)amelioration of triglyceride and ceraminde accumulation in the liver,leg, breast muscle, and heart; d) suppression of the tissuepro-inflammatory IL-1β production and plasma IL-6 concentration; e)cardiac protection and enhanced cardiac function through theup-regulation of the phosphorylation of STAT-3 (signal transducer andactivator of transcription 3) in the heart; f) suppression of theinfiltration of immune cells into the heart; g) decreasing the incidenceof ascites; h) decreasing the incidence of cardiovascular problems; i)decreasing the incidence of cardiac morbidities (dilation, pericardialeffusion, rupture); j) decreasing the amount of irregular incidence ofECG patterns; k) decreasing the occurrence of arrhythmias; l)ameliorating cardiac fibrosis; m) ameliorating chronic systemicinflammation; and n) ameliorating cardiac cell apoptosis; and o)improved insulin resistance and/or blood glucose regulation.
 8. Acomposition, nutraceutical, pharmaceutical composition or premixaccording to claim 1 for the use in improving impaired glucose clearanceand insulin sensitivity
 9. A method of treating human polycystic ovarysyndrome, ameliorating a symptom of polycystic ovary syndrome, ordelaying the onset of polycystic ovary syndrome in a person at risk ofdeveloping the syndrome, comprising: administering to a person who haspolycystic ovary syndrome or who is at risk of developing polycysticovary syndrome an effective amount of a combination comprising:25-hydroxyvitamin D3, Vitamin C, Vitamin E, and one or more carotenoidsselected from the group consisting of: lycopene, astaxanthin,cryptoxanthin, beta-carotene, lutein, zeaxanthin and canthaxanthin. 10.A method of conferring a cardiovascular benefit comprising administeringto a person in need there of an effective amount of a combinationcomprising: 25-hydroxyvitamin D3, Vitamin C, Vitamin E, and one or morecarotenoids selected from the group consisting of: lycopene,astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin andcanthaxanthin; and the cardiovascular benefit is at least one benefitselected from the group consisting of: a) clearance of non-esterifiedfatty acids; b) amelioration of plasma dyslipidemia (triglycerides,sphingomyelin, and ceramide); c) amelioration of triglyceride andceraminde accumulation in the liver, leg, breast muscle, and heart; d)suppression of the tissue pro-inflammatory IL-1β production and plasmaIL-6 concentration; e) cardiac protection and enhanced cardiac functionthrough the up-regulation of the phosphorylation of STAT-3 (signaltransducer and activator of transcription 3) in the heart; f)suppression of the infiltration of immune cells into the heart; g)decreasing the incidence of ascites; h) decreasing the incidence ofcardiovascular problems; i) decreasing the incidence of cardiacmorbidities (dilation, pericardial effusion, rupture); j) decreasing theamount of irregular incidence of ECG patterns; k) decreasing theoccurrence of arrhythmias; l) ameliorating cardiac fibrosis; m)ameliorating chronic systemic inflammation; n) ameliorating cardiac cellapoptosis; and o) improved insulin resistance and/or blood glucoseregulation.
 11. A method of ameliorating weight gain in a hyperphagicperson comprising administering an effective amount of a combinationcomprising: 25-hydroxyvitamin D3, Vitamin C, Vitamin E, and one or morecarotenoids selected from the group consisting of: lycopene,astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin andcanthaxanthin.
 12. A method for improving impaired glucose clearanceand/or insulin sensitivity comprising administering an effective amountof a combination comprising 25-hydroxyvitamin D3, Vitamin C, Vitamin E,and one or more carotenoids selected from the group consisting of:lycopene, astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthinand canthaxanthin to a person in need thereof.